1. Field of the Invention
This invention relates to polyamides which bind to pre-determined sites of the minor groove of double-stranded DNA and have an α-amino acid domain (“positive patch”) capable of inhibiting the activity of major groove DNA-binding proteins.
2. Background of the Invention
Polyamides containing N-methylpyrrole (Py) and N-methylimidazole (Im) amino acids bind to predetermined sequences in the minor groove of DNA with affinities and specificities comparable to naturally occurring DNA binding proteins (Trauger, et al. (1996) Nature 382, 559-561; Swalley, et al. (1997) J. Am. Chem. Soc. 119, 6953-6961; Turner, et al. (1997) J. Am. Chem. Soc. 119, 7636-7644). Sequence specificity is determined by a code of oriented side-by-side pairings of the Py and Im amino (Wade, et al. (1992) J. Am. Chem. Soc. 114, 8783-8794; Mrksich, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 7586-7590; Wade, et al. (1993) Biochemistry 32, 11385-11389; Mrksich, et al. (1993) J. Am. Chem. Soc. 115, 2572-2576; White, et al. (1997) Chem. Biol. 4, 569-578; White, et al. (1997) J. Am. Chem. Soc. 119, 8756-8765). An Im/Py pairing targets a G·C base pair, while Py/Im pair recognizes C·G. The Py/Py pair is degenerate and targets both A·T and T·A base pairs (Pelton, et al. (1989) Proc. Natl. Acad. Sci. USA 86, 5723-5727; Chen, et al. (1994) Nature Struct. Biol. 1, 169-175; White, et al. (1996) Biochemistry 35, 12532-12537). The validity of the pairing rules for ligand design is supported by a variety of polyamide structural motifs which have been characterized by footprinting, affinity cleaving, 2-D NMR, and x-ray methods. The Py/Py pair is degenerate and targets both A·T and T·A base pairs. Polyamides have been found to be cell permeable and to inhibit transcription factor binding and expression of a designated gene (Gottesfeld, et al. (1997) Nature 387, 202-205; Nealy, et al. (1997) J. Mol. Biol. in press). Py/Im polyamides offer a potentially general approach for gene regulation, provided that efficient inhibition of DNA-binding can be achieved for a variety of transcription factors.
Several approaches for the development of synthetic ligands which interfere with protein-DNA recognition have been reported. Oligodeoxyribonucleotides which recognize the major groove of double-helical DNA via triple-helix formation bind to a broad range of sequences with high affinity and specificity (Moser, et al. (1987) Science 238, 645-650; Thuong, et al. (1993) Angew. Chem. Int. Ed. Engl. 32, 666-690). Although oligonucleotides and their analogs have been shown to disrupt protein-DNA binding (Maher, et al. (1992) Biochemistry 31, 70-81; Duval-Valentin, et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 504-508; Nielsen, P. E. (1997) Chem. Eur. J. 3, 505-508), the triple-helix approach is limited to purine tracts and suffers from poor cellular uptake. There are a few examples of carbohydrate-based ligands which interfere with protein-DNA recognition, but oligosaccharides cannot currently recognize a broad range of DNA sequences (Ho, et al. (1994) Proc. Natl. Acad. Sci. USA 91, 9203-9207; Liu, et al. (1996) Proc. Natl. Acad. Sci. USA 93, 940-944). Analogs of distamycin (PyPyPy) appended with multiple cationic substituents have been found to inhibit protein binding. Rational design of tripyrrole peptides that complex with DNA by both selective minor-groove binding and electrostatic interaction with the phosphate backbone. (Bruice (1992) Proc. Natl. Acad. Sci. USA 89, 1700-1704; Chiang, et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2811-2816; Bruice, et al. (1997) Bioorg. Med. Chem. 5, 685-692). Based on these encouraging results, we wished to identify similar charged residues which could be appended to a Py/Im polyamide via linear solid phase synthesis and would not compromise polyamide binding specificity.
Proteins use a diverse structural library to recognize their target sequences (Steitz, T. A. (1990) Quart. Rev. Biophys. 23, 205-280). Proteins such as TBP bind exclusively in the minor groove (Kim, et al. (1993) Nature 365, 512-520), others, such as GCN4 Oakley, M. G. & Dervan, P. B. (1990) Structural motif of the GCN4 DNA binding domain characterized by affinity cleaving (Oakley, et al. (1990) Science 248, 847-850; Ellenberger, et al. (1992) Cell 71, 1223-1237; König, et al. (1993) J. Mol. Biol. 233, 139-154), bind exclusively in the major groove, and certain proteins such as Hin recombinase recognize both grooves (Sluka, et al. (1990) Biochemistry 29, 6551-6561; Feng, et al. (1994) Science 263, 348-355). Polyamides have been found to interfere with protein-DNA recognition in cases where contacts in the minor groove are important for protein-DNA binding affinity. For example, within the nine zinc-finger protein TFIIIA, fingers 4 and 6 bind in or across the minor groove and are required for high affinity binding (Ka=5×109 M−1). An eight-ring hairpin polyamide (Ka=3×1010 M−1) targeted to the minor groove contact region of finger 4 has been recently found to efficiently inhibit protein binding.
X-Ray crystallography studies reveal that DNA bound by a 4-ring homodimeric polyamide is unaltered from its natural B-form structure, with all polyamide/DNA contacts confined to the minor groove (Kielkopf, et al. Nature Struct. Biol., in press). Polyamides have been shown to bind simultaneously with ligands that exclusively occupy the major groove (Oakley, et al. (1992). Biochemistry 31, 10969-10975; Park, et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6653-6657). For example, an 8-ring hairpin polyamide and a recombinant protein containing only the three amino-terminal zinc fingers of TFIIIA which are in the major groove were found to co-occupy the TFIIIA binding site. Similarly, the three-ring homodimer ImPyPy bound simultaneously with the bZIP protein GCN4 (226-281).
Intrinsic DNA curvature and protein induced DNA bending are also involved in the regulation of gene transcription, replication initiation, and other processes (Perez-Martin, J., et al. (1994) Microbiological Reviews 58, 268-290; Polaczek, et al. (1997), submitted). DNA is an inherently flexible polymer and neutral backbone analogs of DNA curve, where rigidity is maintained in natural DNA by coulombic repulsion between phosphates on the same strand Strass, et al. (1994) Science 266, 1829-1834; Manning, G. S. (1983) Biopolymers 22, 689-729). Sequence-dependent curvature of DNA is caused both by differential solvation in the minor groove and differential base stacking leading to alteration of roll and tilt values (Dlakic, et a (1996) J. Biological Chemistry 271, 17911-17919; Bolshoy, et al. (1991) Proc. Natl. Acad. Sci., USA 88, 2312-2316).
Proteins and other ligands that bend DNA alter the stacking of the bases by intercalation of hydrophobic groups, alter the effective Debye length of the surface through charge screening, or bend through energetic compensation for tight binding events. An example of a protein that seems to work through all three mechanisms in bending DNA >160 degrees is integration host factor (IHF) (Rice, et al. (1996) Cell 87,1295-1306). Previously, it has been shown that artificial sequence specific DNA bending ligands can be designed that utilize bidentate tight binding third strand oligonucleotides to constrict the intervening duplex and bend DNA (Liberles, et al. (1996) Proc. Natl. Acad. Sci., USA 93, 9510-4; Akiyama, et al. (1996) Proc. Natl. Acad. Sci., USA 93, 1212212127; Akiyama, et al. (1996) J. Biological Chemistry 271, 29126-29135; Akiyama, et al. (1997) Biochemistry 36,2307-2315).
Compounds that bind in the minor groove such as distamycin and DAPI have been shown to alter DNA rigidity (Larsson, et al. (1996) J. Physical Chemistry 100, 3252-3263; McCarthy, et al. (1991) Nucleic Acids Research 19, 3421-9; Barcelo, et al. (1991) Biochemistry 30, 4863-73.). While such compounds form few specific contacts and binding is dominated by the positive charge, polyamide analogs of distamycin have been designed that form specific high affinity structures with DNA in the minor groove. In such compounds, sequence specificity is determined by the sequence of side-by-side amino acid pairings, where imidazole (Im) opposite pyrrole (Py) recognizes a GC base pair, Py-Im recognizes CG, Py-Py is degenerate for AT or TA, while Im-Im pairing is disfavored (Wade, et al. (1992) J. Am. Chem. Soc. 114, 87838794; Mrksich, et al. (1992) Proc. Natl. Acad. Sci., USA 89,7586-7590; Wade, et al. (1993) Biochemistry 32, 1138511389; Pelton, et al. (1989) Proc. Natl. Acad. Sci., USA 86, 57235727; Pelton, et al. (1990) J. Am. Chem. Soc. 112,1393-1399). This recognition motif generality has been demonstrated for a large number of sequences and is directly supported by NMR data (Mrksich, et al. (1993) J. Am. Chem. Soc. 115, 2572-2576; Geierstanger, et al. (1994) Biochemistry 33, 3055-3062; Geierstanger, et al. (1993) J. Am. Chem. Soc. 115,4474-4482; Geierstanger, et al. (1994) Science 266, 646-650; Mrksich, et al. (1995) J. Am. Chem. Soc. 117,3325-3332; Mrksich, et al. (1993) J. Am. Chem. Soc. 115, 9892-9899; Dwyer, et al. (1993) J. Am. Chem. Soc. 115, 9900-9906; Mrksich, et al. (1994) J. Am. Chem. Soc. 116, 3663-3664; Mrksich, et al. (1994) J. Am. Chem. Soc. 116, 79837988; Chen, et al. (1994) J. Am. Chem. Soc. 116, 6995-7005; Cho, et al. (1995) Proc. Natl. Acad. Sci., USA 92, 10389-10392).